Field of the Invention
The present invention relates to a washing solution for hybridization-enrichment-capture DNA sequencing libraries, and a washing method thereof.
Description of Related Art
Double-stranded nucleic acid molecules (e.g. DNA, DNA/RNA, RNA/RNA) exist in double-helix structures. The double-helix structure is stabilized by the hydrogen bonds between complementary bases (e.g. A+T/U or G+C) of two strands and the hydrophobic force for base stacking. Complementary base pairing is the central dogma of all processes involving nucleic acids. In a basic hybridization reaction, nucleic acid probes or primers are designed to be able to complementarily bind with target sequences. Hybridization-enrichment-capture DNA sequencing libraries is one application of such principle for targeted sequencing of particular genomic regions or a particular group of genes.
The efficiency and precision of nucleic acid hybridization depend on three factors: (1) conditions of denaturation (i.e. separation); (2) conditions of renaturation (i.e. reannealing); (3) conditions of post-hybridization washing. Once the complementary strands are separated, primers or probes would bind to target nucleic acids during the “renaturation” step. This step sometimes is also called the “hybridization” step. In the course of hybridization-enrichment-capture of DNA sequencing libraries, biotinylated probes which are hybridized with genomic target sequences would bind closely with streptavidin beads and in turn be captured. Afterwards, all unbound or non-specifically bound DNA sequences would be removed through a series of washing steps. The stringency of these washing steps largely decides the specificity of bonds between target sequences and capture probes. DNA duplexes of high complementarity display greater stability under stringent washing conditions than those of low complementarity. As such, raising the stringency of washing steps would facilitate the removal of non-specifically bound probes and genomic DNA sequences.
The stringency of the washing steps depends on three adjustable factors: 1. Temperature: as temperature rises, non-specifically bound probes and genomic DNA sequences become denatured and separated; 2. Salt concentration: as salt concentration drops, non-specifically bound probes and genomic DNA sequences become denatured and separated; 3. Incubation time: as washing time increases, non-specifically bound probes and genomic DNA sequences become denatured and separated. Other factors such as pH value and number of washing would also affect the stringency of the washing steps.